A large body of evidence underscores the importance of transcriptional regulation in many complex eukaryotic processes. The long-term goal of the proposed research is to elucidate the molecular mechanisms of transcription by RNA polymerase II (RNAPII). This proposal incorporates genetic, molecular biological and biochemical approaches to test presented hypotheses regarding both the general mechanism of transcription initiation by S. cerevisiae RNAPII and the conserved coordinated functions of RNAPII and the general transcription factors IIB (TFIIB) and TFIIF during transcription initiation in yeast and humans. In Aim 1. genetic approaches will be used to investigate the roles of, and functional interactions between, TFIIB, TFIIF and RNAPII in the mechanism of start site utilization. In Aim 2. biochemical approaches will be employed to determine the architecture of S. cerevisiae preinitiation complexes (PICs) and to analyze the effects of mutant TFIIB, TFIIF and RNAPII proteins on the network of protein-nucleic acid and protein-protein interactions. One hypothesis to be tested is that the architecture of S. cerevisiae PICs is similar to that of higher eukaryotes and that the ability of S. cerevisiae RNAPII to utilize start sites far downstream from a TATA element is by virtue of an energy-dependent translocation subsequent to PIC formation. In Aim 3, biochemical approaches will be employed to determine the effects of mutant yeast and human TFIIB, TFIIF and RNAPII proteins on i) stabilization of the RNA-DNA hybrid in the polymerase active center and ii) various post-assembly activities of yeast and human PICs. The hypotheses to be tested are i) that mutations in the TFIIB finger and the RNAPII switch 2 region that confer downstream shifts in start site utilization impair the ability of the mutant proteins to stabilize the RNA-DNA hybrid and leads to an increased rate of abortive initiation; ii) that mutations in TFIIF that confer upstream shifts in start site usage impair an interaction between TFIIF and RNAPII that leads to a decreased rate of abortive initiation; and iii)that mutations in the identified conserved residues in TFIIB, TFIIF and switch 2 confer alterations to fundamental mechanistic steps in both the yeast and human transcription systems. The combined results from these studies will provide novel information regarding both the unique aspects of the S. cerevisiae RNAPII transcription machinery and the conserved functions of RNAPII, TFIIB and TFIIF during the productive utilization of a transcription start site in eukaryotes.